Biocatalytic process-development continues to advance toward discovering alternative transformation reactions to synthesize medicinally important molecules such as, β-hydroxy-α-amino acids. These bifunctional building blocks, a subclass of noncanonical amino acids, have two stereocenters and are valuable in the natural product, pharmaceutical, and agrochemical sectors.Here, a 5-methylidene-3,5-dihydro-4H-imidazol-4-one (MIO)-dependent phenylalanine aminomutase from Taxus canadensis (TcPAM) was repurposed to irreversibly biocatalyze an intermolecular amino group transfer regioselectively from (2S)-styryl-α-alanine to ring-substituted racemic trans-3-arylglycidates (i.e., cinnamate epoxides) to make the corresponding arylserines. The reaction scope of MIO-aminomutases, which primarily catalyze the reversible interconversion of α- and β-amino acids, can also catalyze the hydroamination of arylacrylates to form α- and β-amino acids. Here, we explore glycidates as a new class of substrates for the MIO-aminomutase family of enzymes. Racemic trans-3-arylglycidates were regio- and stereoselectively aminated to produce a mixture of anti-arylserine enantiomers predominantly. 3-Arylglycidates usually prefer nucleophilic attack at the benzylic Cβ due to stabilization of the partial cationic properties (δ+) at the Cβ by the aryl ring, a stabilization that does not occur at Cα. TcPAM catalysis, however, inverted this inherent nucleophilic regioselectivity by aminating at the Cα of trans-3-arylglycidates to make arylserine predominantly (97%) over arylisoserine (3%). From among twelve substrates, the aminomutase ring-opened 3'-Cl-phenylglycidate to 3'-Cl-phenylserine 140 times faster than it opened the 4'-Cl-isomer, which was turned over slowest among all epoxides tested. GC/MS analysis of chiral auxiliary derivatives of the biocatalyzed arylserine analogues showed that each product mixture contained (2S)+(2R)-anti and (2S)+(2R)-syn pairs with the anti-isomers predominating (~90:10 dr). Integrating the vicinal proton signals in the 1H-NMR spectrum of the biocatalyzed arylserines and calculating the chemical shift difference (Δδ) between the anti and syn proton signals confirmed the diastereomeric ratios and relative stereochemistries. Application of a (2S)-threonine aldolase from E. coli further established the absolute stereochemistry of the chiral derivatives of the diastereomeric biocatalyzed products. The 2R:2S ratio for the biocatalyzed anti-isomers was highest (88:12) for 3'-NO2-phenylserine and lowest (66:34) for 4'-F-phenylserine. This showed that the stereospecificity of TcPAM is, in part, directed by the substituent-type on the arylglycidate analogue.We also synthesized enantiopure 3-phenylglycidates and incubated them separately with TcPAM. The absolute configurations of the biocatalyzed anti-phenylserine (major) and phenylisoserine (minor) were evaluated to gain insights on the substrate specificity and selectivity of TcPAM for aminating 3-phenylglycidate enantiomers. TcPAM converted (2S,3R)-3-phenylglycidate to (2S)-anti-phenylserine predominantly (89%) and (2R,3S)-3-phenylglycidate to (2R)-anti-phenylserine (88%) over their antipodes with inversion of configuration at Cα in each case. Both glycidate enantiomers formed a small amount (~10%) of the syn-phenylserine with retention of configuration at the Cα. TcPAM had a slight preference toward (2S,3R)-3-phenylglycidate, which was turned over (k_cat = 0.3 min-1) 1.5 times faster than the (2R,3S)-glycidate (k_cat = 0.2 min-1). The kinetics data showed that the amination of arylglycidate process follows a two-substrate ping-pong mechanism with competitive inhibition by the epoxide substrate at higher concentration.
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